engleski

Host fitness effects of aminoglycoside resistance 16S rRNA G1405 and A1408 methyltransferases from clinical pathogens and natural antibiotic producers

16S rRNA modification by the G1405 and A1408 methyltransferases (MTases) is a protection mechanism in Actinomycetes that naturally produce various aminoglycosides, which prevents newly synthesized antibiotic to bind to the ribosome. Unfortunately, these enzymes are found in a growing number of clinical pathogens where they cause a high-level resistance and pose an imminent threat to the successful use of aminoglycoside antibiotics. G1405 and A1408 make part of evolutionary conserved decoding site in the small ribosomal subunit, which is heavily modified by other housekeeping MTases. We and others have found that G1405 and A1408 MTases interfere with housekeeping MTases that act on the neighbouring nucleotides. The effect on the E. coli fitness cost has been examined only for three enzymes found in clinical strains in two independent studies based on different experimental systems and results were divergent. We extended the fitness cost study to 8 enzymes found in both clinical strains and antibiotic producers. We examined how constitutively expressed MTases affect the growth of E. coli by measuring the generation time in exponential phase as well as growth rate in growth competition experiments with the parental strain. We tested the translation accuracy by measuring the CUG initiation, UGA read through and -1 frameshifting in beta-galactosidase reporter assay. The complete analysis was extended to the E. coli strain with inactivated gene for the housekeeping MTase RsmF, whose action was found to be impaired by aminoglycoside resistance enzymes in previous studies. Our results show that all aminoglycoside enzymes show negative effect on the growth rate of the host in growth competition experiments, but there is a notable difference between the enzymes from the clinical strains vs. the ones from the producers. While bacteria expressing MTases from clinical strains completely disappear from the mixed culture after 5–6 days of growth, bacteria expressing enzymes from antibiotic producers still make 20% of the mixed culture even after 10 days. On the other hand, we observed that translation accuracy results vary between the strains expressing different aminoglycoside resistance MTases and depending on the presence of the housekeeping MTase. This suggests that even though the enzymes belong to the same family and have a very similar crystal structure, their binding to the ribosome and their catalytic mechanisms may show subtle differences responsible for the versatile fitness cost effects. It is therefore of great importance to further analyse these differences and consider them for the successful design of new drugs that would overcome aminoglycoside resistance.

aminoglycoside resistance methyltransferase; fitness cost

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